Cd146 fitc

To confirm the phenotype of the undifferentiated DPPSC, FACS analysis was performed. The following fluorochrome labelled monoclonal antibodies were used: CD105-FITC (R&D Systems), CD29-PE (R&D Systems), CD146-FITC (BD Pharmingen), CD45-PE (BD Pharmingen), NANOG-FITC and OCT3/4-FITC (R&D Systems). To analyze the control samples, different IgG isotypes coupled to PE and FITC fluorochromes (BD Pharmingen) were used. The cells were suspended in PBS with 2% FBS and were incubated for 45 min at 4 °C in the absence of light. Subsequently, the cells were washed twice with 2% FBS-PBS and centrifuged for 6 min at 1800 rpm, thereby removing any residual fluorochrome to avoid false positive results. The pellets were re-suspended in volumes between 300 and 600 μl (depending on the number of cells) of PBS with 2% FBS. The flow cytometry measurements were made using a FACS cytometer (FACS Calibur, BD Biosciences) and analyzed with WinMDI 2.8 software. To detect and exclude nonspecific unions and auto fluorescence, at least 5x10⁵ cells were used for each sample.

Analysis of cell‐surface antigen expression levels was performed by incubation of the isolated cells. Antihuman antibodies included CD90–Allophycocyanin (APC), CD34‐Fluorescein isothiocyanate (FITC), CD45‐Peridinin chlorophyll protein (PerCP) (Miltenyi Biotec, Germany), CD14‐FITC, CD166‐Phycoerythrin (PE), CD44‐FITC, CD146‐FITC, HLA‐DR‐PerCP, CD73‐PE (BD Biosciences), and STRO1‐pure (secondary anti‐IgM PE conjugated IgG; Santa Cruz Biotechnology Inc., USA).BD FACSCalibur™ was employed for flow cytometric analysis.

Synovium was digested in a solution of 3 mg/mL collagenase (Sigma-Aldrich Japan, Tokyo, Japan) at 37 °C. After 3 h, the digested cells were filtered through a 70-μm cell strainer (Greiner Bio-One GmbH, Kremsmunster, Austria). The cells from six donors were harvested using a cell-dissociation buffer. Cells were suspended in HBSS at a density of 5 × 10⁵ cells/mL and stained for 30 min on ice with the antibodies. For cell isolation, cells were stained with CD31-PE-Cy7 (BD), CD45-PE-Cy7 (BD), CD235a-PE-Cy7 (BD), CD55-FITC (Miltenyi Biotec), CD90-PE (BD) and CD271-APC (Miltenyi Biotec) were used at day 0. Flow cytometric isolation of cell surface antigens were performed by a double-laser Aria 2 system (BD). For cell surface analysis, cells were stained with CD31-FITC (BD), CD45-FITC (BD), CD44-APC-H7 (BD), CD73-BV421 (BD), CD90-PE (BD), CD105-PerCP-Cy5.5 (BD), CD55-PE (BD), CD271-APC (Miltenyi Biotec), CD140b-PerCP-Cy5.5 (BD) and CD146-FITC (BD) at passage 3. Flow cytometric analysis of cell surface antigens was performed by a triple-laser FACS Verse system (BD). These data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). Flow cytometric analyses were also performed for expanded cells at passage 3.

For evaluation of cell surface marker antigens on the cells separated after MACS, cells were stained with CD146‐FITC (BD Biosciences), CD105‐PE, and CD90‐FITC (Miltenyi Biotec, Germany) based on the protocols for immuofluorescent staining.
To confirm neural differentiation, immunostaining was performed for ß‐tubulin III (Promega cat number: G7121) as a specific antibody against neurons. Briefly, the cells were fixed with 4% PFA in +4°C for 20 min, after that they were permeabilized with 0.01% Triton for 10 min, following by adding blocking solution (5% BSA) for preventing nonspecific binding of antibody. The cells were washed three times with 1X PBS, then they were incubated with primary antibody at concentration of 1:500 for 1 hr in room temperature; after washing primary antibody with 1X PBS, secondary antibody Alexa flour 568 goat antimouse (Abcam cat number #ab175473) was added to the cells and was kept for 45 min in room temperature. The stained cells were observed with Olympus microscope (BX53).